Solution and surface effects on plasma fibronectin structure

As assessed by electron microscopy, the reported shape of the plasma fibronectin molecule ranges from that of a compact particle to an elongated, rod-like structure. In this study, we evaluated the effects of solution and surface conditions on fibronectin shape. Freeze-dried, unstained human plasma fibronectin molecules deposited at pH 7.0-7.4 onto carbon films and examined by scanning transmission electron microscopy appeared relatively compact and pleiomorphic, with approximate average dimensions of 24 nm X 16 nm. Negatively stained molecules also had a similar shape but revealed greater detail in that we observed irregular, yarn-like structures. Glutaraldehyde-induced intramolecular cross-linking did not alter the appearance of plasma fibronectin. Molecules deposited at pH 2.8, pH 9.3, or after succinylation were less compact than those deposited at neutral pH. In contrast, fibronectin molecules sprayed onto mica surfaces at pH 7, rotary shadowed, and examined by transmission electron microscopy were elongated and nodular with a contour length of 120-130 nm. Sedimentation velocity experiments and electron microscopic observations indicate that fibronectin unfolds when it is succinylated, when the ionic strength is raised at pH 7, or when the pH is adjusted to 9.3 or 2.8. Greater unfolding is observed at pH 2.8 at low ionic strength (less than 0.01) compared with material at that pH in 0.15 M NaCl solution. We conclude that (a) the shape assumed by the fibronectin molecule can be strongly affected by solution conditions and by deposition onto certain surfaces; and that (b) the images of fibronectin seen by scanning transmission electron microscopy at neutral pH on carbon film are representative of molecules in physiologic solution.     

Morphology of sodium deoxycholate-solubilized apolipoprotein B-100 using negative stain and vitreous ice electron microscopy

The primary and secondary structures of apolipoprotein B-100 (apoB-100) are well established. Previous morphological studies have suggested that apoB is a long, flexible, threadlike molecule that encircles the low density lipoprotein (LDL) particle. Several large domain regions of the protein have been observed in frozen hydrated LDL and may be involved in anchoring of the protein to the lipid surface of LDL. Calorimetric studies of sodium deoxycholate (NaDC)-solubilized apoB indicated a similar number of independently melting domains. We therefore undertook a morphological study of NaDC-solubilized apoB-100 using negative stain and vitreous ice cryoelectron microscopy, a nonperturbing preservation technique. Negative staining experiments were performed in two ways: 1) grids were pulled through NaDC-containing buffer surfaces on which monolayers of apoB had been promoted, or 2) apoB molecules were allowed to diffuse onto carbon surfaces of grids that were floated on sample droplets. Vitrified molecules of apoB were obtained by plunging a thin fluid layer of protein adhered to a holey carbon-coated grid into supercooled ethane and by preserving the molecules in liquid nitrogen. The majority of molecules prepared in negative stain and vitreous ice were curved or arced and had alternating thin and thick regions. In negative stain, the apoB molecules lay on the grid perpendicular to the electron beam and had a mean length of 650 A. In vitreous ice the molecules were randomly oriented and their images ranged from 160 to 650 A in length. Vitrified molecules provided visualization of one or two beaded regions. Similar regions were observed in negative stain but the overall thickness was two to three times greater. Some vitrified molecules contained ribbon-like portions.Our study supports previously obtained data on molecule length but suggests that negative staining overestimates molecule width. These first images of vitrified NaDC-solubilized apoB-100 confirm the long, flexible, beaded thread morphology of the molecule and support the unique potential of this technique when coupled with proper molecule orientation and antibody labeling to correlate the tertiary structure of apoB seen in the intact particle with that of the isolated molecule.     

Trimerization of cell adhesion molecule L1 mimics clustered L1 expression on the cell surface: influence on L1-ligand interactions and on promotion of neurite outgrowth

Several studies indicate that cell adhesion molecules have to be clustered on the cell surface to engage in adhesive functions. We investigated adhesive functions of clustered versus monomeric L1 extracellular parts in vitro to distinguish how clustering affects ligand binding and promotion of neurite outgrowth. Trimeric L1 was recombinantly expressed and covalently assembled by the cartilage matrix protein's coiled-coil domain. Trimeric L1 has an apparent molecular mass of approximately 380 kDa in the nonreduced form and approximately 130 kDa in the reduced form. Rotary shadowing electron micrographs of trimeric L1 revealed a rod-like shape terminating in three globular domains. Monomeric L1 assumes a horseshoe shape of domains Ig I-IV followed by a rod-like structure consisting of Ig V and VI and fibronectin type III 1-5. Circular dichroism measurements showed that the secondary structure consists of beta-sheets. Trimeric L1 binds to itself, to monomeric L1, to laminin-1, and to alpha5beta1 integrin in a concentration-dependent manner. In contrast, binding of monomeric L1 could only be saturated with itself but not with laminin-1 and with alpha5beta1 integrin. Promotion of neurite outgrowth from PC12 cells cultured on adsorbed trimeric L1 was increased by 100%, whereas on monomeric L1 the increase was only 50% over the control value. Promotion of neurite outgrowth from PC12 cells was specifically inhibited in a concentration-dependent manner by a polyclonal antibody against L1. These findings show that clustering of only three extracellular domains increases considerably L1's binding affinity to different ligands and enhances neurite outgrowth, suggesting that adhesive functions of L1 on the cell surface depend on cluster formation.     

Molecular shape and self-association of vinculin and metavinculin

Vinculin, a 130,000-dalton protein localized to adhesion plaques, and metavinculin, a 150,-000 dalton protein closely related to vinculin, have been studied using rotary shadowing and electron microscopy. Both proteins have globular head regions attached to rod-shaped tail domains. Vinculin and metavinculin also both form complexes consisting of four to six individual molecules. These multimers are formed by head-to-head as well as tail-to-tail interactions. Talin, another protein which has been localized to adhesion plaques and binds to both vinculin and metavinculin, has also been investigated using shadowing techniques. Talin is an elongated, flexible molecule in high ionic strength buffers, as shown here by rotary shadowing and negative stain electron microscopy.     

A soluble VE-cadherin fragment forms 2D arrays of dimers upon binding to a lipid monolayer

A high concentration of cadherin molecules at cell-cell adhesion sites is believed to be essential for generating strong intercellular junctions. In order to determine the interactions of cadherin domains involved in the early stages of lateral cluster formation on the cell surface, a recombinant fragment encompassing the first four domains of human VE-cadherin with a His-tag at the C terminus (VE-EC1-4-His) was produced. Two-dimensional crystals of VE-EC1-4-His were formed at the air-water interface using conventional lipids modified to contain a Ni(2+)-chelating group, which provides a specific site for interaction with the polyhistidine tag. The VE-EC1-4-His was monomeric at the concentration employed for crystal formation; however, the crystals exhibited a p2 symmetry and the presence of cis-dimer interactions between symmetry-related molecules. The VE-EC1-4-His molecules in the crystalline array have a remarkably compact conformation in contrast to the elongated "string of pearls" conformation seen in the hexameric assembly of VE-EC1-4-His in solution, and as seen in the crystal structure of C-cadherin. These results indicate that VE-cadherin can exist in at least two oligomeric states with different interactions between domains and can adopt highly different conformational states. We suggest that the compact cis-dimeric state may occur on isolated cells and that the compact form may serve to protect the molecule from degradation. As previously proposed we suppose that the trans-hexameric form is involved in intercellular adhesion.     

Concerning the axial rotational flexibility of the Fab regions of immunoglobulin G

By electron microscopy, we have observed immunocomplexes with both negative stain and in amorphous ice using monoclonal antibodies directed against one of the 24 subunits of scorpion haemocyanin. A copy of this subunit occurs at each of the corners of the square-shaped haemocyanin molecule. Three distinct orientations of adjacent haemocyanin molecules may be observed in immunocomplex pairs or chains using both the above-mentioned methods. These observations, coupled with low-resolution computer simulations of immunocomplex formation, argue strongly in favour of the existence of a considerable degree of rotational flexibility within the IgG molecule and around the long axis of the Fab arms, as was suggested by previous observations with negative stain. We find that the arms can rotate by up to 180 degrees with respect to the Fc region.     

Flexibility and extensibility in the titin molecule: analysis of electron microscope data.

Muscle elasticity derives directly from titin extensibility, which stems from three distinct types of spring-like behaviour of the I-band portion of the molecule. With progressively greater forces and sarcomere lengths, the molecule straightens and then unfolds, first in the PEVK-region and then in individual immunoglobulin domains. Here, we report quantitative analysis of flexibility and extensibility in isolated titin molecules visualized by electron microscopy. Conformations displayed by molecules dried on a substrate vary from a random coil to rod-like, demonstrating highly flexible and easily deformable tertiary structure. The particular conformation observed depends on the "wettability" of the substrate during specimen preparation: higher wettability favours coiled conformations, whereas lower wettability results in more extended molecules. Extension is shown to occur during liquid dewetting. Statistical methods of conformational analysis applied to a population of coiled molecules gave an average persistence length 13.5(+/-4.5) nm. The close correspondence of this value to an earlier one from light-scattering studies confirms that conformations observed by microscopy closely reflected the equilibrium conformation in solution. Analysis of hydrodynamic forces exerted during dewetting also indicates that the force causing straightening of the molecules and extension of the PEVK-region is in the picoNewton range, whereas unfolding of the immunoglobulin and fibronectin domains may require forces about tenfold higher. The microscope data directly illustrate the relationship between titin conformation and the magnitude of applied force. They also suggest the presence of torsional stiffness in the molecule, which may affect considerations of elasticity.     

Structure and hydrodynamic properties of plectin molecules

Plectin is a cytoskeletal, high molecular weight protein of widespread and abundant occurrence in cultured cells and tissues. To study its molecular structure, the protein was purified from rat glioma C6 cells and subjected to chemical and biophysical analyses. Plectin's polypeptide chains have an apparent molecular weight of 300,000, as shown by one-dimensional sodium dodecyl sulfate/polyacrylamide electrophoresis. Cross-linking of non-denatured plectin in solution with dimethyl suberimidate and electrophoretic analyses on sodium dodecyl sulfate/agarose gels revealed that the predominant soluble plectin species was a molecule of 1200 X 10(3) Mr consisting of four 300 X 10(3) Mr polypeptide chains. Hydrodynamic properties of plectin in solution were obtained by sedimentation velocity centrifugation and high-pressure liquid chromatography analysis yielding a sedimentation coefficient of 10 S and a Stokes radius of 27 nm. The high f/fmin ratio of 4.0 indicated a very elongated shape of plectin molecules and an axial ratio of about 50. Shadowing and negative staining electron microscopy of plectin molecules revealed multiple domains: a rigid rod of 184 nm in length and 2 nm in diameter, and two globular heads of 9 nm diameter at each end of the rod. Circular dichroism spectra suggested a composition of 30% alpha-helix, 9% beta-structure and 61% random coil or aperiodic structure. The rod-like shape, the alpha-helix content as well as the thermal transition within a midpoint of 45 degrees C and the transition enthalpy (168 kJ/mol) of secondary structure suggested a double-stranded, alpha-helical coiled coil rod domain. Based on the available data, we favor a model of native plectin as a dumb-bell-like association of four 300 X 10(3) Mr polypeptide chains. Electron microscopy and turbidity measurements showed that plectin molecules self-associate into various oligomeric states in solutions of nearly physiological ionic strength. These interactions apparently involved the globular end domains of the molecule. Given its rigidity and elongated shape, and its tendency towards self-association, plectin may well be an interlinking element of the cytoskeleton that may also form a network of its own.     

Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake

A binary complex of the ammonia channel Amt1 from Methanococcus jannaschii and its cognate P(II) signalling protein GlnK1 has been produced and characterized. Complex formation is prevented specifically by the effector molecules Mg-ATP and 2-ketoglutarate. Single-particle electron microscopy of the complex shows that GlnK1 binds on the cytoplasmic side of Amt1. Three high-resolution X-ray structures of GlnK1 indicate that the functionally important T-loop has an extended, flexible conformation in the absence of Mg-ATP, but assumes a compact, tightly folded conformation upon Mg-ATP binding, which in turn creates a 2-ketoglutarate-binding site. We propose a regulatory mechanism by which nitrogen uptake is controlled by the binding of both effector molecules to GlnK1. At normal effector levels, a 2-ketoglutarate molecule binding at the apex of the compact T-loop would prevent complex formation, ensuring uninhibited ammonia uptake. At low levels of Mg-ATP, the extended loops would seal the ammonia channels in the complex. Binding of both effector molecules to P(II) signalling proteins may thus represent an effective feedback mechanism for regulating ammonium uptake through the membrane.     

Crystal structure of the actin-binding domain of alpha-actinin-4 Lys255Glu mutant implicated in focal segmental glomerulosclerosis

Mutations in α-actinin-4 have been linked to familial focal segmental glomerulosclerosis (FSGS), a common renal disorder in humans, and produce an apparent increase in the actin-binding affinity of α-actinin-4 in vitro. One of the mutations, in particular, Lys255Glu, falls in the middle of the actin-binding interface of the actin-binding domain (ABD). The ABD consists of tandem calponin homology (CH) domains (CH1 and CH2). The crystal structures of most ABDs display a compact conformation, characterized by extensive inter-CH interactions. However, the conformation of F-actin-bound ABDs is unsettled. Some electron microscopy studies find that the compact conformation is preserved upon binding to F-actin, whereas other studies suggest that the CHs separate and the ABD becomes extended. The Lys255Glu mutation in CH2 is significant in this regard since it removes a crucial inter-CH interaction with Trp147 of CH1, thought to stabilize the compact conformation. Together, the increased actin-binding affinity and the removal of this important inter-CH contact suggested that the Lys255Glu mutation might facilitate the transition toward the open ABD conformation proposed by some of the electron microscopy studies. However, the crystal structure of the ABD of α-actinin-4 Lys255Glu mutant described here displays the canonical compact conformation. Furthermore, the sedimentation coefficients by analytical ultracentrifugation of wild-type and FSGS mutant ABDs (Lys255Glu, Ser262Pro, and Thr259Ile) are nearly identical (2.50 ± 0.03 S) and are in good agreement with the theoretical value calculated from the crystal structure (2.382 S), implying that the compact conformation is retained in solution. The absence of a structural change suggests that the compact ABD conformation observed in the majority of the structures is highly stable and is preserved in solution, even in FSGS mutant ABDs.     

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.     

Membrane association induces a conformational change in the Ebola virus matrix protein

The matrix protein VP40 from Ebola virus is targeted to the plasma membrane, where it is thought to induce assembly and budding of virions through its association with the lipid bilayer. Ebola virus VP40 is expressed as a monomeric molecule in solution, consisting of two loosely associated domains. Here we show that a C-terminal truncation of seven residues destabilizes the monomeric closed conformation and induces spontaneous hexamerization in solution, as indicated by chemical cross-linking and electron microscopy. Three-dimensional reconstruction of electron microscopy images shows ring-like structures consisting of the N-terminal domain along with evidence for flexibly attached C-terminal domains. In vitro destabilization of the monomer by urea treatment results in similar hexameric molecules in solution. In addition, we demonstrate that membrane association of wild-type VP40 also induces the conformational switch from monomeric to hexameric molecules that may form the building blocks for initiation of virus assembly and budding. Such a conformational change induced by bilayer targeting may be a common feature of many viral matrix proteins and its potential inhibition may result in new anti-viral therapies.     

3D Structure of fish muscle myosin filaments

Myosin filaments isolated from goldfish (Carassius auratus) muscle under relaxing conditions and viewed in negative stain by electron microscopy have been subjected to 3D helical reconstruction to provide details of the myosin head arrangement in relaxed muscle. Previous X-ray diffraction studies of fish muscle (plaice) myosin filaments have suggested that the heads project a long way from the filament surface rather than lying down flat and that heads in a single myosin molecule tend to interact with each other rather than with heads from adjacent molecules. Evidence has also been presented that the head tilt is away from the M-band. Here we seek to confirm these conclusions using a totally independent method. By using 3D helical reconstruction of isolated myosin filaments the known perturbation of the head array in vertebrate muscles was inevitably averaged out. The 3D reconstruction was therefore compared with the X-ray model after it too had been helically averaged. The resulting images showed the same characteristic features: heads projecting out from the filament backbone to high radius and the motor domains at higher radius and further away from the M-band than the light-chain-binding neck domains (lever arms) of the heads.     

Regulation of Adhesion by Flexible Ectodomains of IgCAMs

To perform their diverse biological functions the adhesion activities of the cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) might be regulated by local clustering, proteolytical shedding of their ectodomains or rapid recycling to and from the plasma membrane. Another form of regulation of adhesion might be obtained through flexible ectodomains of IgCAMs which adopt distinct conformations and which in turn modulate their adhesion activity. Here, we discuss variations in the conformation of the extracellular domains of CEACAM1 and CAR that might influence their binding and signaling activities. Furthermore, we concentrate on alternative splicing of single domains and short segments in the extracellular regions of L1 subfamily members that might affect the organization of the N-terminal located Ig-like domains. In particular, we discuss variations of the linker sequence between Ig-like domains 2 and 3 (D2 and D3) that is required for the horseshoe conformation.     

Outline structure of the human L1 cell adhesion molecule and the sites where mutations cause neurological disorders.

The L1 cell adhesion molecule has six domains homologous to members of the immunoglobulin superfamily and five homologous to fibronectin type III domains. We determined the outline structure of the L1 domains by showing that they have, at the key sites that determine conformation, residues similar to those in proteins of known structure. The outline structure describes the relative positions of residues, the major secondary structures and residue solvent accessibility. We use the outline structure to investigate the likely effects of 22 mutations that cause neurological diseases. The mutations are not randomly distributed but cluster in a few regions of the structure. They can be divided into those that act mainly by changing conformation or denaturing their domain and those that alter its surface properties.     

Nucleotide-dependent shape changes in the reverse direction motor, myosin VI

We have studied the shape of myosin VI, the actin minus-end directed motor, by negative stain and metal shadow electron microscopy. Single particle processing was used to make two-dimensional averages of the stain images, which greatly increases the clarity and allows detailed comparisons with crystal structures. A total of 169,964 particle images were obtained from two different constructs in six different states (four nucleotide states and with and without Ca²⁺). The shape of truncated apo myosin VI was very similar to the apo crystal structure, with the lever arm bent strongly backward and around the motor domain. In the full-length molecule, the C-terminal part of the tail has an additional bend taking it back across the motor domain, which may reflect a regulated state. Addition of ATP, ADP, or ATP-γS resulted in a large change, straightening the molecule from the bent shape and swinging the lever by ∼140°. Although these nucleotides would not be expected to produce the pre-powerstroke state, myosin VI in their presence was most similar to the truncated crystal structure with bound ADP-VO₄, which is thought to show the pre-powerstroke shape. The nucleotide data were therefore substantially different from expectation based on crystal structures. The full-length molecule was almost completely monomeric; only ∼1% were dimers, joined through the ends of the tail. Addition of calcium ions appeared to result in release of the second calmodulin light chain. In negatively stained molecules there was little indication of extended α-helical structure in the tail, but molecules viewed by metal shadowing had a tail ∼3× longer, 29 vs. 9 nm, part of which is likely to be a single α-helix.     

Use of negative stain and single-particle image processing to explore dynamic properties of flexible macromolecules

Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unstained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used negative staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of negative staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor.     

The compact domain conformation of human Glu-plasminogen in solution.

A complete understanding of the accelerating mechanisms of plasminogen activation and fibrinolysis necessarily requires structural information on the conformational forms of plasminogen. Given the absence of high-resolution structural data on plasminogen the use of lower resolution approaches has been adopted. Two such approaches have previously indicated a compact conformation of Glu-plasminogen (Tranqui, L., Prandini, M., and Chapel, A. (1979) Biol. Cellulaire, 34, 39-42; Bányai, L. and Patthy, L. (1985) Biochim. Biophys. Acta, 832, 224-227) whereas a third has suggested a fairly extended conformation (Mangel, W., Lin, B. and Ramakrishnan, V. (1990) Science, 248, 69-73). Native Glu-plasminogen has been investigated using small-angle X-ray scattering (SAXS) experiments. It is concluded that this molecule in solution is compact (radius of gyration, RG 3.05 +/- 0.02 nm and maximum intramolecular distance, Im 9.1 +/- 0.3 nm) and that the data are consistent with the right-handed spiral structure observed using electron microscopy by Tranqui et al. (1979). A spiral structure of native plasminogen would have important implications for the conformational response of plasminogen to fibrin and concomitant stimulation of plasminogen activation.     

Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy

An important part of characterizing any protein molecule is to determine its size and shape. Sedimentation and gel filtration are hydrodynamic techniques that can be used for this medium resolution structural analysis. This review collects a number of simple calculations that are useful for thinking about protein structure at the nanometer level. Readers are reminded that the Perrin equation is generally not a valid approach to determine the shape of proteins. Instead, a simple guideline is presented, based on the measured sedimentation coefficient and a calculated maximum S, to estimate if a protein is globular or elongated. It is recalled that a gel filtration column fractionates proteins on the basis of their Stokes radius, not molecular weight. The molecular weight can be determined by combining gradient sedimentation and gel filtration, techniques available in most biochemistry laboratories, as originally proposed by Siegel and Monte. Finally, rotary shadowing and negative stain electron microscopy are powerful techniques for resolving the size and shape of single protein molecules and complexes at the nanometer level. A combination of hydrodynamics and electron microscopy is especially powerful.     

Crystal structure of the actin-binding domain of alpha-actinin 1: evaluating two competing actin-binding models

Alpha-actinin belongs to the spectrin family of actin crosslinking and bundling proteins that function as key regulators of cell motility, morphology and adhesion. The actin-binding domain (ABD) of these proteins consists of two consecutive calponin homology (CH) domains. Electron microscopy studies on ABDs appear to support two competing actin-binding models, extended and compact, whereas the crystal structures typically display a compact conformation. We have determined the 1.7A resolution structure of the ABD of alpha-actinin 1, a ubiquitously expressed isoform. The structure displays the classical compact conformation. We evaluated the two binding models by surface conservation analysis. The results show a conserved surface that spans both domains and corresponds to two previously identified actin-binding sites (ABS2 and ABS3). A third, and probably less important site, ABS1, is mostly buried in the compact conformation. However, a thorough examination of existing structures suggests a weak and semi-polar binding interface between the two CHs, leaving open the possibility of domain reorientation or opening. Our results are consistent with a two-step binding mechanism in which the ABD interacts first in the compact form observed in the structures, and then transitions toward a higher affinity state, possibly through minor rearrangement of the domains.